This study aimed to evaluate the antibacterial activity of zinc oxide nanoparticles (ZnO-NPs) on Staphylococcus aureus (S. aureus) isolated from layer chickens using minimum inhibitory concentration test (MIC). A total number of 120-layer chicken samples from hock joints and livers were collected under aseptic conditions from diseased and freshly dead layer chickens from different private farms in Assiut Governorate. From these 120 samples, we isolated 29 isolates of Staphylococci with a prevalence rate of 24.16%. Nineteen (65.5 %) of these 29 isolates were coagulase negative and ten (34.5 %) were coagulase positive. By PCR, the 10 coagulase positive strains (CoPS), were confirmed to be S. aureus as they have (clfA gene), while 6 isolates only were Methicillin Resistant Staphylococcus Aureus (MRSA) as they demonstrated mecA gene. Nine isolates of S. aureus demonstrated variable antibiotic sensitivity patterns in MIC test against ZnO-NPs and some selected antibiotics. ZnO-NPs showed the best antibacterial efficiency against most isolates as compared to all tested antibiotics.